Protocols


Screen (pdf version)
ScreenEx Vivo Macrophage Screen for Control of Viral Infection
Posted On02/18/2010 12:24 PM
AuthorCeline Eidenschenk, Sungyong Won
Science WriterEva Marie Y. Moresco
Background
In this screen, thioglycolate-elicited peritoneal macrophages from ENU-mutagenized G3 mice are used to discover components important for the early control of mouse cytomegalovirus (MCMV), adenovirus, influenza virus, and Rift Valley Fever virus (RVFV) infections. Each virus used in this screen is titered so that only a small percentage of target cells is infected. After an incubation period during which the virus is replicated and released to infect new cells (except here in the case of adenovirus), the total numbers of virus-infected cells are compared between C57BL/6J control and G3 strains (Figure 1). Strains for which a significantly higher number of cells are infected relative to wild type are identified as potential mutants. In parallel with this screen, the supernatants from virus-infected macrophages are analyzed for tumor necrosis factor (TNF) and type I interferon (IFN) content (Figure 2). These cytokines are induced by most viral infections, and provide another indication of viral control. Compared to in vivo screens in which susceptible animals may die from viral infection (see MCMV Susceptibility and Resistance Screen and In Vivo RVFV Susceptibility Screen), the use of macrophages facilitates more rapid recovery of the mutant strain, since the animal is kept alive and only macrophages are taken for testing. However, because the full complement of cell types found in the intact organism is lacking, the macrophage screen cannot reveal all genes required for viral control.
 
Macrophages are critical in the defense against infection, functioning as phagocytes as well as interacting with and stimulating other components of the immune system. They are important factors in the pathogenesis and outcome of viral infections, and are restrictive for some viruses, such as herpes simplex virus and vesicular stomatitis virus (1).  In the case of MCMV, peritoneal macrophages are permissive for its infection, support MCMV replication, and release plaque-forming virus into the culture medium to infect other cells (2;3). Latently infected mice harbor MCMV in peritoneal macrophages for extended periods of time, from which virus can be recovered upon macrophage activation (2;4). It has been proposed that exacerbation of latent MCMV infections in vivo may be triggered by the activation of macrophages (2).
 
Adenoviruses are a frequent cause of acute upper respiratory tract infections, as well as eye, gastrointestinal tract and bladder infections. In the respiratory tract, adenoviruses infect epithelial cells, causing an early, rapid recruitment of neutrophils, macrophages and monocytes which initiate the inflammatory response. Macrophages do not express the 46-kDa cell surface receptor CAR (coxsackie/adenovirus receptor) for the adenovirus fiber knob, which mediates high-affinity binding of the virion to the host cell, and thus internalize virus 100- to 1000-fold less efficiently than lung epithelial cells (5).  Instead, adenoviruses utilize CD46 as their primary attachment receptor on macrophages (6). Adenoviruses have been intensively studied as a tool for gene transfer to a variety of cell types. This screen utilizes the human adenovirus 5 serotype expressing the fiber protein from serotype 16 (Ad5-F16), which is able to infect mouse macrophages.  As this adenoviral vector is non-replicating, an abnormally elevated fraction of cells expressing the GFP tag must reflect enhanced infectivity or greater permissiveness of the cells in expressing the transfected marker rather than propagation of the vector.
 
Peritoneal macrophages may also be infected by influenza, although with lower efficiency compared to alveolar macrophages that normally encounter the virus in vivo (7). Once infected, at least one round of viral replication occurs followed by apoptosis of the infected macrophages, and their phagocytosis by surrounding uninfected macrophages and dendritic cells (8-10). Phagocytosis of infected cells by alveolar macrophages is an important mechanism limiting the propagation of influenza in vivo (9). For this screen, a mouse-adapted human influenza A virus (PR8 strain) is used (see Influenza Resistance Screen).
 
Mice with mutations in known genes were used to test the requirement for interferon (IFN) and Toll-like receptor (TLR) signaling in the control of MCMV, influenza and adenovirus by macrophages (Figure1 and Table 1). Relative to C57BL/6J control macrophages, increased percentages of Ifnar-/- and Stat1domino/domino macrophages are infected by each of the viruses, demonstrating a requirement for type I IFN signaling in viral control, consistent with published data (11-14). In contrast to type I IFN signaling, IFN-γ signaling is dispensable for control of all three viruses by macrophages. For MCMV, this result is in contrast to studies indicating a role for IFN-γ in macrophage activation during MCMV infection in vivo (15). However, the situation is complex, as IFN-γ-mediated macrophage activation has been shown to be negatively regulated by type I IFN (16). IFN-γ signaling is important for adenovirus control in respiratory airway epithelial cells (17), but its role in macrophages has not been reported. IFN-γ is not required for the control of influenza A virus in vivo (18;19). Interestingly, signaling through TLRs is not required for control of MCMV, influenza, or adenovirus in macrophages, as demonstrated by the infection of similar percentages of cells from wild type mice or mutants deficient in TLR3, TLR7, TLR9, Unc93b1 (3d), or MyD88 (pococurante) (Table 1). These findings suggest redundant macrophage sensing mechanisms for these viruses, and/or differing requirements for IFN and TLR signaling in macrophages versus other cell types.
 
RVFV (genus Phlebovirus, family Bunyaviridae), a mosquito-borne, hepatotropic virus, causes epidemics in ruminants of sub-Saharan Africa but also infects humans, where it typically results in flu-like illness, but may cause myalgia, hemorrhagic fever, ocular disease, encephalitis, and death (20). RVFV has been shown to infect the human monocytic cell line U937, and the mouse macrophage cell line J774.1 (21;22). The NSs protein of RVFV blocks transcription by the transcription factor TFIIH, resulting in suppression of type I IFN production and consequently, innate immune responses (23;24).  IFN-α and IFN-γ therapies prevent or greatly suppress RVFV disease in rhesus monkeys (25;26). Accordingly, peritoneal macrophages lacking type I IFN signaling as a result of mutations in either IFNAR1 (macro-1), STAT1 (domino), or IRF1 (Endeka), cannot control RVFV infections as efficiently as wild type cells (Figure 3 and Table 1). In addition, TLR signaling is dispensable for control of RVFV in macrophages (Table 1). Two recombinant viruses are used in screening: a mutagen-attenuated RVFV vaccine strain containing the complete genome (arMP-12) (27), and a mutant virus derived from arMP-12 in which the gene encoding the viral NSs protein has been replaced with the coding sequence for GFP (arMP-12-delNSs/GFP).
 
Reagents and Solutions
Brewer’s thioglycolate medium, 4%
4% (w/v) Brewer’s thioglycolate medium powder (BBL Microbiology Systems, Cockeysville, MD) is added to distilled water pre-warmed to 37°C. Solution is autoclaved to sterilize and stored away from light.
 
PEC recovery solution
Hepes-buffered saline solution (Gibco, Invitrogen, Carlsbad, CA )
5% (v/v) heat-inactivated fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA)
200 IU/mL penicillin (Gibco)
200 mg/mL streptomycin (Gibco)
 
PEC medium
Dulbecco’s modified eagle medium (Mediatech Inc., Herndon, VA)
5% (v/v) heat-inactivated fetal bovine serum
200 IU/mL penicillin
200 mg/mL streptomycin
 
FACS buffer
Phosphate buffered saline (PBS)
5% (v/v) heat-inactivated fetal bovine serum
 
Perm/Wash Buffer (BD Biosciences, #554723)
 
Perm/Fix Solution (BD Biosciences, #554722)
 
α-hemagglutinin antibody and appropriate FITC-conjugated secondary antibody
 
α-RVFV nucleoprotein (N) rabbit polyclonal antibody (28), and α-rabbit FITC-conjugated secondary antibody
Method
Peritoneal exudate cell (PEC) isolation
1. Four days prior to PEC isolation, 5mL syringes filled with Brewer’s thioglycolate medium are used to inject mice intraperitoneally with 1.5-2mL through a 25-gauge needle.
2. Immediately prior to isolation, mice are anaesthetized under isofluorane vapour (2-5% v/v, 2% O2).
3. 5mL syringes filled with sterile PBS are used to recover PECs by lavage through an 18-gauge needle. Once obtained, exudate is added to 5mL of PEC recovery solution in a 15mL conical tube, and stored on ice.
4. Tubes containing exudate are centrifuged for 5 minutes at 1500 rpm in a tabletop centrifuge, and supernatant is replaced with 1mL of PEC medium. Pelleted cells are resuspended by pipeting, and a 20μL aliquot is taken for cell enumeration.
5. The concentration of each cell sample is adjusted to 1x106 cells/mL using PEC medium, and 100μL of each sample (1x105 cells) is added per well to a tissue culture-treated 96 well flat-bottomed plate. Each virus to be tested requires one well of cells. Plates are incubated at 37oC/5%CO2 in a humidified incubator for 24 hours to allow cells to adhere to the plate.
Viral infections and FACS analysis
The MCMV-GFP strain, generated by insertion of the GFP gene into the MCMV bacterial artificial chromosome plasmid pSM3fr (29), is from Dr. Chris Benedict (La Jolla Institute of Allergy and Immunology, La Jolla, CA). The virus is propagated on mouse embryonic fibroblasts and purified as described (30). Titres are determined by plaque assay on mouse embryonic fibroblasts.
 
Ad5-F16-GFP is from Dr. Glen Nemerow (The Scripps Research Institute, La Jolla, CA), and was constructed as described (31). The virus is grown in 293 cells, purified by CsCl gradient, and particle number is calculated as described (31;32).
 
Influenza virus is grown in chicken eggs and titred using a standard hemagglutination assay (Influenza Resistance Screen).
 
The RVFV strain arMP-12 was recovered from cDNA through a reverse genetics system (33). arMP-12-delNSs/GFP was generated from the parental arMP-12 strain. The viruses are propagated and titred by plaque assay in Vero E6 cells as described (22).
 
1. Viruses are diluted in PEC medium and added to the prepared PECs at the following dosages:
a. MCMV-GFP (MOI 1)
b. Ad5-F16-GFP (104 particles/cell)
c. influenza (1.5 hemagglutination units per well)
d. arMP-12 (MOI 1 and MOI 0.01)
e. arMP-12-delNSs/GFP (MOI 1 and MOI 0.01)
2. PECs are incubated with the viruses for the following times:
a. MCMV-GFP, 24 hours
b. Ad5-F16-GFP, 72 hours
c. influenza, 24 hours
d. arMP-12, 16 hours
e. arMP-12-delNSs/GFP, 48 hours
3. The number of virus-infected cells is determined by flow cytometry.
For MCMV-GFP-, Ad5-F16-GFP- and arMP-12-delNSs/GFP-infected cells:
a. Invert the plate to empty, and then wash each well twice with 100μL PBS.
b. Add 50μL trypsin to each well, and return the plate to the incubator for 10-15 minutes.
c. Transfer the contents of each well into individual FACS tubes, and add 100μL of FACS buffer.
d. Count the number of GFP+ cells by FACS.
 
For influenza-infected cells:
a. Invert the plate to empty, and then wash each well twice with 100μL PBS.
b. Add 50μL trypsin to each well, and return the plate to the incubator for 10-15 minutes.
c. Transfer the contents of each well to a fresh 96 well plate.
d. Add 100μL of FACS buffer to each well, and then centrifuge the plate.
e. Invert the plate to empty, and then add 50μL Perm/Fix Solution to each well. Incubate for 20 minutes at 4°C.
f. Add 150μL Perm/Wash Buffer to each well, and then centrifuge the plate.
g. Invert the plate to empty, and rinse with 100μL Perm/Wash Buffer. Centrifuge and invert the plate to empty.
h. Add 50μL of α-HA antibody, diluted 1:200 in Perm/Wash Buffer, to each well, and incubate for 30 minutes (or overnight) at 4°C.
i. Add 100μL of Perm/Wash Buffer to each well, and then centrifuge the plate.
j. Rinse each well again with 100μL Perm/Wash Buffer, as in step g.
k. Add 50μL of FITC-conjugated secondary antibody diluted in Perm/Wash Buffer to each well, and incubate for 30 minutes (or overnight) at 4°C.
l. Add 100μL of Perm/Wash Buffer to each well, and then centrifuge the plate.
m. Invert the plate to empty, and resuspend the cells in 150μL FACS buffer.
n. Count the number of HA+ cells by FACS.
 
For RVFV-infected cells:
a. Invert the plate to empty, and then wash each well twice with 100μL PBS.
b. Add 50μL trypsin to each well, and return the plate to the incubator for 10-15 minutes.
c. Transfer the contents of each well to a fresh 96 well plate.
d. Add 100μL of FACS buffer to each well, and then centrifuge the plate.
e. Invert the plate to empty, and then add 50μL Perm/Fix Solution to each well. Incubate for 20 minutes at 4°C.
f. Add 150μL Perm/Wash Buffer to each well, and then centrifuge the plate.
g. Invert the plate to empty, and rinse with 100μL Perm/Wash Buffer. Centrifuge and invert the plate to empty.
h. Add 50μL of α-RVFV nucleoprotein (N) antibody, diluted 1:1000 in Perm/Wash Buffer, to each well, and incubate for 30 minutes (or overnight) at 4°C.
i. Add 100μL of Perm/Wash Buffer to each well, and then centrifuge the plate.
j. Rinse each well again with 100μL Perm/Wash Buffer, as in step g.
k. Add 50μL of FITC-conjugated secondary antibody diluted in Perm/Wash Buffer to each well, and incubate for 30 minutes (or overnight) at 4°C.
l. Add 100μL of Perm/Wash Buffer to each well, and then centrifuge the plate.
m. Invert the plate to empty, and resuspend the cells in 150μL FACS buffer.
n. Count the number of RVFV N+ cells by FACS.
 
Alleles Identified
A9649
atchoum
macro-1
macro-2
References